GSM7473674: Inoculation_3day_replication1; Manihot esculenta; RNA-Seq
The total RNA from inoculated leaves was purified by use of the Plant RNA reagent (Thermofisher) following RNeasy Plant Mini Kit (QIAGEN). A 0.5 mL of Plant RNA reagent was added to about 0.1 g of fresh weight of finely powdered leaf tissue that was crushed by a Multi-Beads Shocker (2,000 rpm, 15 s) and its mixture was mixed gently. After incubating for 5 minutes at room temperature, the supernatant was collected by centrifuging at 12,000 × g for 2 minutes at room temperature. The supernatant was transferred to a clean RNase-free tube. A 0.1 mL of 5 M NaCl was added to supernatant and tube was mixed by tapping. A 0.3 mL of chloroform was added to the supernatant and mixed thoroughly by inverting. The supernatant was collected by centrifugation at 12,000 x g for 10 minutes at 4°C to separate the phases. The upper aqueous phase was transferred to a clean RNase-free tube. After an equal volume of isopropyl alcohol was added to the collected aqueous phase, the mixture was incubated at room temperature for 10 minutes. After centrifuging the mixture at 12,000 x g for 10 minutes at 4°C, the precipitation was washed by 1.0 mL of 75% ethanol. After the solubilization of the precipitates in 40 μL of RNase-free water, the solution was treated with DNase I (10 U)(Takara) and RNase inhibitor (40 U) (Nippongene) in 50 μL of reaction at 37°C for 20 min. The 50 μL of solution was added to the 350 uL of RLT buffer in RNeasy Plant Mini Kit (QIAGEN) and the RNA preparation was performed as described by manual procedure. The total RNA quality was evaluated by electrophoresis with the bioanalyzer system (Agilent, USA). The total RNA was stored at -80°C until further use. We used 500 ng of the total RNA of each sample for library preparation. The library was prepared by following the Lasy-Seq (Kamitani et al. 2019) version 1.1 protocol (https://sites.google.com/view/lasy-seq/).